Molecular diversity in Lactucaspeciesusing Isozymes and RAPD Markers

Understanding of the molecular basis of genetic diversity in Lactucaaccessions is substantial for the management, improvementand ecient uses of Lactuca accessions. Therefore, this workaimed to evaluate molecular diversity among twenty-six accessions of Lactuca species usingisozymes and RAPD analyses. The polymorphic percentages were 87.09%and 100% in isozymes and RAPD analyses respectively, indicating a high genetic variation within and among Lactuca species. The number of alleles were higher in the wild species compared to the cultivated species, reecting a reduction in the richness of alleles in the cultivated species due to domestication that caused a reduction in genetic diversity to meet the demand for high crop productivity.Isozymes and RAPD clustering dendrogrames: (1) separated,L. sativa accessions in more than one cluster conrming their polyphyletic origin; (2)collected the accessions of L. vimineain one cluster revealed its homogeneity; and (3) divided the accessions of L.saligna in two clusters varied in the number of alleles, particularly “A” form. The corresponding analysis associated the accessions of the wild species based on the alleles “B”of the tested isozymes and the cultivated species on alleles “A” and “C”, suggesting that: (1) allele “B” might be the primitive form of these loci that can tolerate the environmental stresses which prevails in the habitats of the wild species, and (2) “A” and “C” could be the derived forms. These results are of great interest for the management of Lactuca germplasm and for future breeding programs of lettuce.


Introduction
The genus Lactuca L. is a member of subtribe Lactucinae, tribe Lactuceae, that belongs to family Lactuceae (Asteraceae) (Güzel et al. 2018). It includes annual, biennial or perennial herbs, rarely shrubs with abundant latex. The species ofLactuca distributed mainly in warm and temperate geographical regions of the world (van Herwijnen and Manning 2017). However, the continents Asia and Africa are the center of the diversity of the genus. (Doležalová et al., 2002;Lebeda et al. 2004).
The primary gene pool of L. sativa L. is represented by a worldwide spread L. serriola, further L. aculeata, L. scarioloides, L. altaica,L. azerbaijancia, L. georgica originating in Asia and L. dregeananative to South Africa Page 3/13 Isozymes have been used as a reproducible marker in assessing the amount and distribution of genetic variability and systematic relationshipswithin and between Lactuca spp. (Kesseli and Michelmore 1986;Dziechciarková et al. 2004). Theyexhibited that the genetic variability was lower in intra-species compared with inter-species and suggestedthat the origin of L.sativawaspolyphyletic. The systematic relationship in Lactuca spp. was investigated using isozymes: (1)confirming the genetic closeness between L. aculeata and L. sativa as they are members of L. serriola complex, (2) revealing a wide genetic distance between L. saligna and L. virosa, and (3) identifying L. serriola, L. saligna, L. virosa, and landrace L. sativa as distinct entities (Roux et al. 1985;Kesseli and Michelmore 1986).
The use of molecular markers is also important in assessing the level of genetic diversity and in defining the genetic relationship between and within species, populations and accessions. Randomly Amplified Polymorphic DNA marker (RAPD) is one of the class of DNA markers that has received maximum attention in investigating the genetic variability because it is less expensive, less technical, fast and involves no radioactivity and hybridization. It was used in establishing the differences among lines of apparently closely related accessions in germplasm collections of butterhead and crisphead lettuce (Waycott and Fort 1994).It wasalso successfully used in identifying between twelve lettuce varieties (Yamamoto et al. 1994). Furthermore, it was used to investigate the genetic variation and interspecific relationship among cultivars of L. sativa var. capitate, exhibiting a monophyletic cluster with similarity coefficient values ranging from 0.61 to 0.98 (Yoo and Jang 2003).
Genetic resources collections of Lactuca species are poorly characterized because they are made up of large groups of numbered accessions of Lactuca that lack descriptive and (or) pedigree . So, little information is available on the genetic variation among different accessions of Lactuca representing different regions in the world. Therefore, the aim of this research was to use isozymes and DNA markers to evaluate the genetic variability and genetic relationships within and betweenLactuca species.

Plant materials
The experimental material was obtained from the CGN (Centre for Genetic Resources, Wageningen, The .(Netherlands). Itconsists of 26 accessions of Lactuca spp. (Table 1 Protein extraction, electrophoresis and activity staining Isozymes crude extracts were prepared by macerating 20 mg young leaves of fifteen days old seedlings with 1 mL of extraction buffer consisted of 0.05M sodium phosphate buffer (pH 7.2), 20% v/v glycerol, 14mM2mercaptoethanol and 0.05% v/v triton X-100 (Manchenko 1994).The clear supernatant was applied directly on 7% PAGE at 4°C in a Mini Protean III unit (BioRad, California, USA), under a constant current of 100 mA for 5 to 6hr, until the tracking dye had moved 5 to 7cm from the cathodal end. The gels were subjected to activity staining for Phosphorylase, Catalase, α-Esterase and β-Esterase isozymes following the protocols of Pasteur et al. (1988). Phosphorylase gels were stained in solution formed of 10 mM I 2 mixed with14 mM KI after incubation in 100 ml solution of 0.1M sodium phosphate buffer (pH 5.1) at 37 0 C for 3 to 5 h, developing white bands on a dark blue background. The chromatic or light brown bands appeared at the bottom of the gels were amylase Page 4/13 bands. The gels of catalase were stained by immersing in 1:1 mixture of solutions 2% potassium ferricyanide and 2% ferric chloride after incubation in a solution of 3% H 2 O 2 for about 15 min. The gels were then washed and gently agitated for a few minutes in water. Yellow bands of Catalase activity appeared on a blue-green background. The gels of α and -esterases were incubated at 37°C for 15 min in 100 ml staining solution consisted of 0.05 M phosphate buffer (pH 7.2) containing 1% α or β naphthyl acetate for α and -esterases respectively and 50 mg Fast Blue RR until brown colored bands appeared. The stained gels were photographed as quickly as possible and stored in 3% acetic acid. At least 5 and generally 10 plants per accession were .examined for isozyme patterns DNA extraction forRAPD analysis g young leaves of fifteen days old seedlings were ground with a pestle in liquid nitrogen. The ground sample 0.5 was suspended in 1 ml preheated CTAB buffer (1.4 MNaCl, 0.2 % 2-mercaptoethanol, 100 mMTris-Cl and 20 mM EDTA) at 65ºC for one hour. The suspended solution was centrifuge at 1000 rpm and the supernatant was mixed with 0.5 ml of 24:1 chloroform: isomyl, then centrifuge at 14000 rpm (Doyle and Doyle 1990). To precipitate the nucleic acid, the aqueous layer was mixed with ice cold isopropanol, then incubated overnight at -20ºC and centrifuged at 14000 rpm. The supernatant was discarded and the pellet was carefully washed twice with cold .70% ethanol, dried at room temperature and re-suspended in 100 µl of sterile de-ionized distilled water RAPD amplification Genomic DNAs of the studied accessions were amplified in 25µl reaction mixture, containing 20ng DNA, 0.5 unit Taq polymerase (Sigma-Aldrich, St. Louis, MO, USA), 0.2 mM PCR Nucleotide Mix (Boehringer Mannheim, Tubingen, Germany), 0.5 µM RAPD primers, 5µl amplification buffer, 1.5µl of MgCl 2 and 9.75µl of distilled H 2 O (Williams et al.1990). Amplification was performed for 45 cycles using a Biometera Uno thermal cycler (SPW Industrial, Laguna Hills, CA, USA): One cycle at 95 0 C for 3 minutes, then 44 cycles at 92 0 C for 2minutes, 37 0 C for 1minute and 72 0 C for2minutes. The reactions were finally run at 72 0 C for 10 min and further incubated on ice, at 4 0 C. The primers were chosen based on their ability to produce reproducible amplification patterns ( Table 2). The amplification DNAs were separated by electrophoresis on 2% agarose in 50X Tris-Acetate EDTA buffer consisted of 242g Tris-base, 57.1 ml Glacial acetic acid and 100 ml EDTA (0.5 M pH 8.0).

Data analysis
The isozymes and RAPD bands were scored as "0" for presence and "1' for absence for a band at a particular locus in each accessions, which were transformed into a binary character matrix. The binary character matrix of each marker was subjected to multivariate analysis (correspondence analysis and cluster analysis) usingthe software package "PAST", Version 4.02, Natural History Museum, University of Oslo, 1999-2020. The cluster .(analyseswere performed using the unweighted pair-group method with arithmetic mean (UPGMA
The number of alleles ranged from 17 in L.virosa CGN05145 from France to 27 in L. sativax L.serriola group Oilseed CGN05115 from Egypt with an average 20.8 and the mean number of alleles per locus ranged from 0.55 to 0.87with an average 0.67 ( Table 2).The mean frequency of allelesof the 26 accessions was 0.64. It was higher in cultivated species (0.52) compared to the wild ones (0.48). The lowest allele frequency (0.07) was observed for the allele CAT-1A in the accessions of L.perennis.
The clustering dendrogram of the isozymes data gave two main groups (G1 and G2) at genetic distances 2.25 is directly associated with the degree of divergence or similarity among them in respect to their isozymes characters.

RAPD analysis
Among 18 random primers tested in this study, 5 primers generated reproducible bands. A total of 186 polymorphic bands were identified using the five primers. Maximum and minimum percentages of polymorphic bands were observed by primers OPA3 (46 fragments) and OPA1 (31 fragments)respectively. The average percentage of polymorphic bands was 100 % (Table 3). The average size of DNA fragments ranged between 33bp and 600bp ( Figure 3). The fragment with DNA size of: 33 bp is unique marker for L. sativa x L. In the present study, the estimationof genetic diversity with isozyme markers showed high polymorphism between the examined accessions (87.09%) which was attributed to outcrossing of the majority of the species and infrequent interspecific hybridization (Jemelková, et al. 2018).It also showed that the polymorphism between L. sativa accessions (74.2%) was not with same magnitude of the polymorphism between the wild species, The highest number of alleles in the wild species (31) compared with the cultivated species L. sativa (27) reflected a reduction in the richness of alleles due to domestication which was reported to modify agronomic phenotypes and genetic signature of the domesticated species, resulted in the reduction in genetic diversity to meet the demand for high crop productivity and crop uniformity in the field and the marketplace (Dempewolf et al. 2017;Zhang et al. 2017). The variation between cultivated and wild species was not only limited to the number of alleles, but also it extended to the mean frequency of alleles which was found to be higher in the cultivated species(0.52) comparing with the wild ones (0.48). This might be attributed to the absence of selection pressure on the wild species, as the existing variations in these species are natural (Das 2011). The allele with low mean allele frequency (0.07) or what was known as rare alleles was observed only forthe allele CAT-1A in L. perennis. The presence of this allele could be due to deleterious mutations or may be due to evolutionary relics (Sammour et al. 2019). The detection of rare allele in combination with high allelic frequency of other loci leads to the conclusion that the studied accessions had wide genetic differentiation.
The separation of L. dregeana with L. virosain the same cluster in isozymes and RAPD cluster analyses confirmed their closely relatedness as they belong to the same section Lactuca (Lebeda and Astley 1999) The distribution of the accessions of L. sativa in more than one clusterin the isozymesand RAPD dendrogramsconfirmed the polyphyletic origin of L. sativa. Furthermore, the clustering of some of the accessions of L. sativawith L.serriola group Oilseed lettuce based on isozymes data and theclustering of the rest of the accessions with L. dregeanaconfirmed that L. serriola and L. dregeanawere members of the primary gene pool of L. sativa (Zohary 1991) and wasconsistent with the work of Kesselli and Michelmore (1986)who also suggested that domestication of L. sativa may be due to the repeated domestication from wild progenitors or may be due to the use of interspecific hybridizations in breeding programmes to introduce characters of interest into cultivated lettuce. However, the prevailing of the allele "B" in the wild species and alleles "A" and "C" in the cultivated species and its progenitors, as it has been shown in the corresponding analysis, indicating that the cultivated species was firstly domesticated from wild progenitors and later breeders used interspecific hybridizations to introduce characters of interest into cultivated lettuce. The prevailing of the allele "B" in wild species and alleles "A" and "C" in cultivated ones also suggested that: (1) allele "B" might be the primitive form of studied alleles that enable the wild species to resist the environmental stresses which prevails in their habitats, and (2) alleles "A" and "C" could be derived forms that evolved as a result of domestication.
The separation of the accessions of L. salignain two clustersin the cluster analysis of the isozymes data was consistent with the works of Cole et al. (1991) and Esawi et al. (2017).The heterogeneity of this species attributed to the difference in the number and types of alleles in the studied accessions. The accessions Lactuca salignaCGN13327 from Greece and Lactucasaligna CGN10883 from Portugal did not have the allele "A" of the loci ACP-2, CAT-2, CAT-4, αEST-2, βEST-3, βEST-4 which characterize the cultivated species, whereas the accession Lactuca salignaCGN13330 from Turkey had these alleles.This lead to infer that the accession from Turkey mightbe subjected toa natural mutation or a sort of unintended domestication.Although Güzel et al. (2018) observed several samples in the field and herbaria belonging to L. viminea that easily fell into two distinct subspecies according to their habits and morphological traits, the clustering dendrograms of isozymes and RAPD analyses separated the accessions of L. vimineain one cluster which was inconsistent with their observation. The variation observed by Güzel et al. (2018) could be attributed ecogeographical conditions in which the accessions of L. vimineawereoriginated; the conditions that cause a significant variation in the phenotypic characters. The clustering dendrogram based on RAPD data indicated a close genetic relationship between L. sativa, L. serriola, L. salignaand L. virosawhich was consistent with the phylogenetic trees based on chloroplast DNA sequence comparison (Wei et al. 2017),chromosomal studies (Matoba et al. 2007), nrITS1 and AFLP fingerprints (Koopman et al. 1998;Koopman et al. 2001). So, L. serriola, L. salignaand L. virosacan be considered important resources for L. sativa breeding.

Conclusion
The polymorphism in the studied accessions were very high either in isozymes or RAPD analyses, exhibiting high genetic variability within and among Lactucaspecies. Thepolymorphism and the number of alleleswere lower in the cultivated species compared to the wild species, reflecting a reduction in the richness of alleles in the cultivated species due to domestication that caused a reduction in genetic diversity of the cultivated species comparing with the wild ones. The separately multivariate analyses of the isozymes and RAPD data confirmed the polyphyletic origin of L. sativa. They also collected the accessions of L. vimineain one cluster revealing the homogeneity of this species and ruling out the previous study that showed that L. viminea has two distinct subspecies. The corresponding analysis associated the accessions of the wild species based on the alleles "B" of the studied isozymes, the form that might enable the wild species to resist to the environmental stresses that prevails in their habitats; and the cultivated species on alleles "A" and "C". This suggested that allele "B" might be the primitive form of the alleles of the loci of the assessed isozymes, and "A" and "C" could be the derived forms. The separation of the accessions of L.saligna in two clusters was due to the variation in the number of alleles, particularly "A" form, suggesting that some accessions could be wild and the other might be subjected to natural mutation or unintended domestication. Accessions of L. sativa x L. serriolagroup Oilseed, L. indicia, L.
saligna were characterized with unique DNA fragments which can be used as markers for identifying these accessions. The presences of the great genetic variation between cultivated lettuce and its wild relatives opened the path for the improvement of the cultivated species. The association between specific forms of alleles and cultivated and wild species still needed deep insights to be manipulated for lettuce improvement. The considerable genetic variation in the accessions of Lactuca saligna, Lactuca virosa and Lactuca indicaopen the door for more detailed studies on big number of accessions cover the distribution range off these species using morphological, biochemical and molecular markers.  Tables   Tables not available with this